| Background and objection:Caries is a common disease in the field of oral medicine, one of the leading causes of teeth loss. Caries destroy the integrality of masticating apparatus, great unfavorable effect on the digestive function, oral facial aesthetics and psychology of patients. Caries is a kind of chronic progressive infectious disease, occurred in tooth hard tissue, under the influence, which mainly because of bacteria. During the decay process, the bacteria and toxins invade dental pulp tissue along the dentin tubule, reparative dentin formed as a protective barrier of pulp tissue to avoid stimulations from bacteria outside. These undifferentiated mesenchymal cells differenting to odontoblast-like cells after stimulated by stimulations, forming reparative dentin to protect pulp tissue is considered to be an important part of innate immune response of dental pulp. And these undifferentiated mesenchymal cells that have the ability of multiple differentiation is dental pulp stem cells (DPSCs).Innate immune response is the first barrier of human body to against pathogens invasion and tissue damage. The human innate immune response is carried out with the process of pattern recognition receptors (pattern recognition receptors, PRRs) specific identify pathogen associated molecular patterns (pathogen associated molecular pattern, PAMPs). Toll-like receptor4is a kind of PRRs can combine with PAMPs specificity, starting the innate immune response to against pathogen invasion. Cell proliferation, migration and differentiation are essences of tissue repair. There have some researchers been reported, TLR agonist can promote cell migration, invasion and release a large number of immunomodulatory factor. Lipopolysaccharide (LPS) stimulation enhances adhesion and migration of MDPC-23cells in a process that requires TLR4. LPS might increase the gene expression of the pro-inflammatory cytokines IL-1β, IL-6and, IL-8in dental follicle cells and induce the release of IL-1CCL7, CCL26and CXCL11in fibroblasts. It has been reported that supernatants of stimulated cultures also increased the migration of immature dendritic cells. These data suggest that TLR4may play a pivotal role in tooth repair and regeneration.At present, the mechanism of TLR4in the innte immune response of dental pulp tissue is not clear. This study compared expression of TLR4in healthy dental pulp tissue and pulp tissue with deep caries. These changes of proliferation, migration and mineralization ability of dental pulp stem cells were observed under the condition of TLR4activated or inhibited to make sure the role of TLR4in the innate immune response of dental pulp stem cells.The paper includes the following five parts: Part1:The location of TLR4in healthy dental pulp tissue and dental pulp tissue affected by deep caries.In this part of experiment, we compared the location of TLR4in healthy dental pulp tissue and dental pulp tissue affected by deep caries.Materials and Methods1. HE stainingHealth (n=3) and deep caries (n=3) human third molars were collected from adults (18-26years of age); the informed written consent of the patients was obtained. Teeth were disinfected strictly before the extraction. Immediately upon extraction, teeth were fixed in4%formalin-buffered solution for4hours. Teeth demineralizations were performed with10%EDTA for4weeks. When demineralization completely, dehydration, transparent and embedding treatment were performed. Paraffin sections were stained with hematoxylin and eosin. Morphology of tooth was observed with a microscope.2. ImmunohistochemistryHealth (n=8) and deep caries (n=12) human third molars were collected from adults (18-26years of age); the informed written consent of the patients was obtained. Immediately upon extraction, each tooth was halved lengthwise using a diamond instrument and a hammer. The dental pulp was then carefully extracted and fixed in4%formalin-buffered solution for4hours. Tissues of health and deep caries tooth were embedded in paraffin and sectioned at4-mm thickness. These sections were incubated with TLR4antibodies (dilution1:100; Abcam) overnight at4℃. After washing with phosphate-buffered saline (PBS, pH7.4), the sections were then incubated with secondary antibody (Boster) for1hour at room temperature. Color development was performed with3,30-diaminobenzidine (DAB)(Boster). Nuclei were counterstained with hematoxylin-eosin. Seal pieces with neutral gum. Morphology of tooth was observed with a microscope.3. ImmunofluorescenceSections were incubated with TLR4antibodies (dilution1:100; Abcam) overnight at4℃. After being washed twice with PBS, sections were subsequently incubated with FITC-conjugated rabbit anti-mouse antibody (1:500; Golden Bridge) for1hour at room temperature in the dark. Nuclei were stained with DAPI (Boster). In the negative control sections, a PBS was substituted for the primary antibody. Sealing pieces and morphology of tooth was observed with a fluorescence microscope.ResultsHE stainting was used to observe the basic morphology of healthy and deep caries dental pulp. Immunohistochemical and immunofluorescent staining were performed to make sure the location of TLR4. Results showed that, dentin tubules of tooth affected with deep caries were damaged by bacteria and a lot of bacteria mass were observed, but there was no significant change in the pulp tissue compared with healthy tooth. TLR4was expressed in the odontoblast layer and areas that co-localize with blood vessels of healthy dental pulp. The distribution of TLR4in the pulp of teeth affected by deep caries was similar to that observed in healthy teeth, but much more positive staining was observed. Positive staining was found in these areas that co-localize with blood vessels in teeth with deep caries. Results indicate that, TLR4may participate in the innate immune response of tooth affected with deep caries.Part2:Culture and identification of hDPSCsIn this part of experiment, hDPSCs were isolated and cultured by combination of explants method and enzymatic separation method. Cells were identified as hDPSCs. Providing a stable cell resource to follow-up experiment.Materials and Methods1. Cell cultureHealth human third molars and health tooth needed extract were collected from adults (18-26years of age), the informed written consent of the patients was obtained. Immediately upon extraction, each tooth was halved lengthwise using a diamond instrument and a hammer. Under aseptic conditions, the health pulps were removed and washed3times with PBS. Minced pulp tissues were digested in a solution of3mg/mL collagenase type â… for10min at37℃. Single-cell suspensions were seeded into6-well plates (Costar) with dulbecco modified eagle medium (Hocolny) containing10%fetal bovine serum(Hocolny),100units/mL penicillin,100mg/mL streptomycin, and50mg/mL ascorbic acid and then incubated at37℃,5%CO2. Cells were grown to confluence and continuously passed at a1:2ratio when confluent. The3to5passage cells were used to perform the follow-up experiment. 2. Flow cytometry analysis1×105-5×105/mL passage3DPSCs were collected and incubated with CD29, CD34, CD45, CD90, CD44and CD106antibody for1h in dark. DPSCs were washed twice and fixed with1mL fixatives. Cell phenotype analysis was performed by flow cytometric analysis according to the manufacturer’s protocol.3. Differentiation potential of DPSCs in vitro.DPSCs of passage3were seeded into6-well plates at a density of2×105cells/well and cultured to80confluence. For the mineralization assay, cells were cultured in mineralized solutionfor3-week induction. The induced cells were stained with1%Alizarin Red. For adipogenic differentiation, cells were cultured in fat induced liquid for3-week and stained with oil red O. For chondrogenic induction, the cells were cultured with cartilage induced liquid for3-week and stained with alcian blue. Morphology of cells was observed with a microscope.4. Growth curve detected with cell count1×104/well passage3DPSCs were seeded into24well plates. The number of DPSCs was counted on1d,3d,5d and7d with the coulter counter,6well each group. Experiment repeat thrice, average date were recorded to make the growth curve of DPSCs.5. Growth curve detected with MTT assayDPSCs were seeded into96-well plates at a density of3×103cells/well. The cell proliferation rate was evaluated with3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) according to the manufacturer’s instructions (Sigma).20μL of MTT (5mg/mL) was added to the culture for an incubation time of4hours. 150μL of dimethyl sulfoxide (DMSO) was added to extract and solubilize the formazan. MTT values for each well were measured pectrophotometrically at490nm. The MTT assay was carried out in1,3,5and7day, and the assay was repeated in triplicate.ResultsWe isolated the DPSCs successfully using the combination of explants method and enzymatic separation method. The morphology of these cells was similar with fibroblast and mesenchymal stem cell, growth curve was "S" type. Results of cell phenotype analysis indicated cells were positive expressed CD29, CD44and CD90(surface makers of mesenchymal stem cell,99.26%,95.17%and99.68%respectively) but negative expressed CD34, CD45and CD106(surface makers of hematopoietic stem cell,0.1%,0.33%and0.32%respectively). Furthermore, DPSCs had an ability to differentiate into other cell types. Mineral node, intracellular fat and cartilage were stained with alizarin red, oil red O and alcian blue respectively.Part3:The expression of TLR4in DPSCs stimulated with LPS and extracts from S. mutans.We simulated the internal environment of dental pulp under the condition of deep caries by stimulated with LPS and extracts from S. mutans. Examing wheather LPS and extracts from S. mutans can activate the expression of TLR4in DPSCs.Materials and Methods1. Sonicated extracts from Streptococcus mutans Streptococcus mutans (UA159) was grown in brain-heart infusion broth in an anaerobic chamber for24hours before harvesting. The bacterial cells were harvested by centrifugation at10,000g for20minutes at4℃and washed twice with PBS, and then disrupted using a sonicator (Rongyan) with30seconds bursts at1minute intervals for60minutes. The supernatant was harvested by centrifugation and filtered with a0.22-μm filter membrane. Protein concentrations in the extracts were determined using a protein-assay kit (Boster). Extract from S.mutans was diluted with DMEM to1mg/mL and stored in-80℃。2. Real-Time PCRDPSCs were treated with1μg/mL LPS and1μg/mL extracts from S. mutans. DPSCs were collected at different time points (0,12,24, and48hours) to evaluate the expression of TLR4messenger RNA (mRNA). Total cellular mRNA was obtained from DPSCs using TRIzol (Takara), according to the manufacturer’s recommendations. Reverse transcription was performed by the PrimeScriptR reagent kit (Takara). The cDNA levels were measured by real-time PCR using the SYBR PremiDimerEraserTM kit (Takara). All genes were analyzed using the Applied Biosystems7500Real-Time PCR System. Each sample was analyzed three times, and the mean values were calculated. The amount of target Cdna (TLR4) relative to β-actin was calculated using the formula2-△△CT.3. Western blotDPSCs were treated with1μg/mL LPS and1μg/mL extracts from S. mutans for24h. The total protein was isolated and quantitated using BCA assay. The protein lysates were separated by SDS-PAGE, and electrophoretically transferred to PVDF membrane. Then, the membrane was incubated with antibodies. The protein bands were visualized on X-ray film using the ECL system.4. ImmunocytochemistryDPSCs of passage3-5were seeded into cover glass at a density of3×103cells/well. Cells were treated with1μg/mL LPS and1μg/mL extracts from S. mutans for24h. Cells were fixed with pure acetone for20min at-20℃, permeabilized with PBS containing0.25%(v/v) Triton X-100for10min. Then, blocked with normal goat serum and incubated with the anti-TLR4primary antibody (Abeam) overnight. After they were rinsed, the immunostaining cells were incubated for1hour with secondary antibody and mounted with anti-fade reagent with DAPI. In the negative-control group, PBS was substituted for the primary antibody. Sealing pieces and morphology of tooth was observed with a fluorescence microscope.ResultsExtract from S.mutans were prepared with a sonicator. After stimulations with LPS and extract from S.mutans, the expression of TLR4mRNA was promoted (LPS: F=852.193, P=0.000; S.mutans:F=1471.990, P=0.000), which was detected by RT-PCR. Results of western blot and immunocytochemical staining indicated the TLR4protein was up-regulated.Part4:LPS and extracts from S. mutans induced biological characteristics changes of DPSCs after the activation of TLR4.This part of experiment discussed the effect of LPS and extracts from S. mutans on perliferation, migration and mineralization abilities of DPSCs.Materials and Methods1. Proliferation detected with MTT assayDPSCs were seeded into96-well plates at a density of3×103cells/well and treated with various doses of LPS or extracts from S. mutans.. The cell proliferation rate was evaluated with3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) according to the manufacturer’s instructions (Sigma).20μL of MTT (5mg/mL) was added to the culture for an incubation time of4hours.150μL of dimethyl sulfoxide (DMSO) was added to extract and solubilize the formazan. MTT values for each well were measured pectrophotometrically at490nm. The MTT assay was carried out in1,3,5and7day, and the assay was repeated in triplicate.2. Scratch testDPSCs of passage3-5were seeded into6-well plates at a density of1×105cells/well, a1-mm wound introduced across each plate using a small tip. The width of the wound was measured by the ruler in the image-processing software to ensure uniformity in the distance to each observation point. DPSCs were treated with various doses of LPS or extracts from S. mutans. Cell migration was observed by microscopy at0h,24h and48h respectively. Experiments were repeated in triplicate.3. Transwell assayDPSCs were suspended in medium without FBS, seeded into the upper chamber of transwell at a density of2×104/well. Then,500μL medium containing10%FBS was added into the lower chamber and treated with various doses of LPS or extracts from S. mutans for24hours at37℃. The cells that migrated to the lower surface of the membrane were fixed and stained with crystal violet. The number of migrated cells was counted in five randomly selected high-power fields (200×), average number of date were obtained. Experiments repeated6times. Morphology of cells was observed with a microscope.4. Mineralization assay.DPSCs of passage3-5were seeded into6-well plates at a density of2×105cells/well and cultured to80%confluence. For the mineralization assay, cells were cultured in mineralized solution and treated with1μg/mL LPS and1μg/mL extracts from S. mutans for3-week induction. The induced cells were stained with1%Alizarin Red. Morphology of cells was observed with a microscope.5. Real-Time PCRCells were cultured in mineralized solution and treated with1μg/mL LPS and1μg/mL extracts from S. mutans for3-week induction. Total cellular mRNA was obtained from DPSCs using TRIzol (Takara), according to the manufacturer’s recommendations. Reverse transcription was performed by the PrimeScriptR reagent kit (Takara). The cDNA levels were measured by real-time PCR using the SYBR PremiDimerEraserTM kit (Takara). All genes were analyzed using the Applied Biosystems7500Real-Time PCR System. Each sample was analyzed three times, and the mean values were calculated. The amount of target cDNA (OCN, BSP and ALP) relative to β-actin was calculated using the formula2-△△CT.Results Both LPS and extracts from S. mutans down-regulate the proliferation of DPSCs (P<0.05), even at a relatively low endotoxin doses. Transwell assay demonstrated that, LPS and extracts S. mutans promoted the migration ability of DPSCs (LPS: F=117.722, P=0.000; S.mutans: F=142.676, P=0.000), scratch test obtained a similar result. However, mineralization ability of DPSCs was inhibited, after the stimulation of LPS and extracts from S. mutans. Compared with control group, mineralized nodules (F=3369.577, P=0.000) and mineralization genes OCN (F=74.446, P=0.000), BSP (F=417.217, P=0.000), ALP (F=60.861, P=0.000) were down-regulated significantly.Part5:The regulation effect on DPSCs biological characteristics of TLR4.Si-TLR4lentivirus has been constructed successfully to detect proliferation, migration and mineralization abilities of DPSCs when the expression of TLR4was down-regulated.Materials and Methods1. Construction of Si-TLR4lenttvirus and cell infectionLentiviral vector inhibiting TLR4gene has been successfully constructed and maintains high expression in DPSCs. The lentiviral vectors is pGLV3/Hl/GFP+Puro, packaging vectors are pGag/Pol, pRev and pVSV-G. Recombinant lentiviral vectors and packaging vectors were then transfected into293T cells. Supernatants containing lentiviruses expressing Si-TLR4were harvested72h after transfection. The lentiviruses were then purified by ultracentrifugation, and the titer of lentiviruses was determined. DPSCs were infected with the lentivirus at different multiplicity of infection (MOI) to make sure the suitable MOI for follow-up experiment.2. Real-Time PCRDPSCs were transfected with groups of Si-TLR4and Si-NC. Total cellular mRNA was obtained from DPSCs using TRIzol (Takara), according to the manufacturer’s recommendations. Reverse transcription was performed by the PrimeScriptR reagent kit (Takara). The cDNA levels were measured by real-time PCR using the SYBR PremiDimerEraserTM kit (Takara). All genes were analyzed using the Applied Biosystems7500Real-Time PCR System. Each sample was analyzed three times, and the mean values were calculated. The amount of target cDNA (TLR4) relative to β-actin was calculated using the formula2-△△CT.3. Western blotDPSCs were transfected with groups of Si-TLR4and Si-NC. The total protein was isolated and quantitated using BCA assay. The protein lysates were separated by SDS-PAGE, and electrophoretically transferred to PVDF membrane. Then, the membrane was incubated with antibodies. The protein bands were visualized on X-ray film using the ECL system.4. Proliferation detected with MTT assayDPSCs transfected with Si-TLR4or Si-NC were seeded into96-well plates at a density of3×103cells/well. The cell proliferation rate was evaluated with3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) according to the manufacturer’s instructions (Sigma).20μL of MTT (5mg/mL) was added to the culture for an incubation time of4hours.150μL of dimethyl sulfoxide (DMSO) was added to extract and solubilize the formazan. MTT values for each well were measured pectrophotometrically at490nm. The MTT assay was carried out in1,3,5and7day, and the assay was repeated in triplicate.5. Transwell assayDPSCs transfected with Si-TLR4or Si-NC were suspended in medium without FBS, seeded into the upper chamber of transwell at a density of2×104/well. Then,500μL medium containing10%FBS was added into the lower chamber and incubated for24hours at37℃. The cells that migrated to the lower surface of the membrane were fixed and stained with crystal violet. The number of migrated cells was counted in five randomly selected high-power fields (200×), average number of date were obtained. Experiments repeated6times.6. Mineralization assay.DPSCs transfected with Si-TLR4or Si-NC were seeded into6-well plates at a density of2×105cells/well and cultured to80%confluence. For the mineralization assay, cells were cultured in mineralized solutionfor3-week induction. The induced cells were stained with1%Alizarin Red. Morphology of cells was observed with a microscope.7. Real-Time PCRDPSCs transfected with Si-TLR4or Si-NC were cultured in mineralized solution for3-week. Total cellular mRNA was obtained from DPSCs using TRIzol (Takara), according to the manufacturer’s recommendations. Reverse transcription was performed by the PrimeScriptR reagent kit (Takara). The cDNA levels were measured by real-time PCR using the SYBR PremiDimerEraserTM kit (Takara). All genes were analyzed using the Applied Biosystems7500Real-Time PCR System. Each sample was analyzed three times, and the mean values were calculated. The amount of target cDNA (OCN, BSP and ALP) relative to β-actin was calculated using the formula2-△△CT.ResultsSi-TLR4lentivirus has been constructed successfully and maintains high expression in DPSCs. RT-PCR results demonstrated DPSCs transfectted with Si-TLR4can inhibited the expression of TLR4mRNA (F=196.427, P=0.000) significantly. Result of western blot showed that the expression of TLR4protein was inhibited. Proliferation (P<0.05) and migration abilities (t=12.734, P=0.000) were down-regulated, mineralized nodules (t=-56.160, P=0.000) and mineralization genes OCN (t=-3.416, P=0.027), BSP (t=-12.344, P=0.000) and ALP (t=-55.815, P=0.000) were up-regulated significantly when expression of TLR4was inhibited.Conclusion1. TLR4was expressed in the odontoblast layer and areas that co-localize with blood vessels of healthy and deep caries dental pulp, but much more positive staining was observed in pulp tissue affected with deep caries.2. DPSCs were isolated and cultured successfully using the combination of explants method and enzymatic separation method. These cells expressed surface makers of mesenchymal stem cell, and have an ability to differentiate into other cell types. 3. TLR4of DPSCs can be activated with stimulations of LPS and extract from S.mutans. The proliferation and mineralization abilities of DPSCs were down-regulated, but the migration ability was promoted after stimulations of LPS and extract from S.mutans.4. Si-TLR4lentivirus has been successfully constructed and maintained high expression in DPSCs. When TLR4was inhibited, the migration and proliferation abilities of DPSCs were down-regulated, but the osteogenic differentiation of DPSCs was promoted. |
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